streptomyces iconiensis sp. nov. and streptomyces smyrnaeus sp. nov., two halotolerant actinomycetes...
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Streptomyces iconiensis sp. nov. and Streptomyces smyrnaeus sp. nov., two halotolerant 1
actinomycetes isolated from Tuz (Salt) Lake and Camalti Saltern in Turkey 2
3
Demet Tatar1, Kiymet Guven
2, Cathrin Spröer
3, Hans-Peter Klenk
3,
Nevzat Sahin
1 4
5
6
1 Department of Biology, Faculty of Art and Science, Ondokuz Mayis University, 55139 7
Kurupelit-Samsun, Turkey. (Author for correspondence: E-mail: [email protected]). 8
2 Anadolu University, Faculty of Science, Biology Department, 26470, Eskişehir. 9
3 Leibniz Institute DSMZ−German Collection of Microorganisms and Cell Cultures GmbH, 10
38124 Braunschweig, Germany 11
Author for correspondence: Nevzat Sahin 12
13
Subject category: New Taxa: Actinobacteria 14
15
Running title: Streptomyces iconiensis s sp. nov. and Streptomyces smyrnaeus sp. nov. 16
17
The GenBank accession number for the 16S rRNA gene sequence of Streptomyces iconiensis 18
BNT558T(= KCTC 29198
T = DSM 42109
T) is KC959223 and Streptomyces smyrnaeus 19
SM3501T(= KCTC 29214
T = DSM 42105
T)
is KF006349. 20
21
Keywords: Streptomycetaceae, Streptomyces iconiensis, Streptomyces smyrnaeus, Polyphasic 22
taxonomy 23
24
25
26
IJSEM Papers in Press. Published June 18, 2014 as doi:10.1099/ijs.0.062216-0
Abstract 27
The taxonomic positions of two novel actinomycetes, designated BNT558T and SM3501
T, 28
were established by using a polyphasic approach. The organisms had chemical and 29
morphological features that were consistent with their classification in the genus 30
Streptomyces. The whole-cell hydrolysates of the two strains contained LL-diaminopimelic 31
acid as the diagnostic diamino acid. The predominant menaquinones were MK-9(H6) and 32
MK-9(H8) for strain BNT558T and MK-9(H8) and MK-9(H6) for strain SM3501
T. Major fatty 33
acids of the strains were anteiso-C15:0, anteiso-C17:0 and iso-C16:0. The polar lipid profiles of 34
strain BNT558T contained diphosphatidylglycerol, phosphatidylethanolamine, 35
phosphatidylinositol, one unidentified glycolipid and one unidentified aminophospholipid, 36
while that of strain SM3501T consisted of diphosphatidylglycerol, phosphatidylglycerol, 37
phosphatidylinositol, phoshphatidylethanolamine and three unidentified atypical aminolipids, 38
one unidentified aminolipid and two unidentified glycolipids. The G+C contents of the 39
genomic DNAs were 70.2 and 69.6 mol % for strains BNT558T and SM3501
T, respectively. 40
16S rRNA gene sequence data supported the classification of the isolates in the genus 41
Streptomyces and showed that they formed two distinct branches within the genus 42
Streptomyces. Based on the almost complete 16S rRNA gene sequences isolate BNT558T was 43
most closely related to Streptomyces albiaxialis NRRL B-24327T and isolate SM3501
T was 44
most closely related to Streptomyces cacaoi subsp. cacaoi NBRC 13860T. DNA-DNA 45
relatedness between each of the isolates and its closest phylogenetic neighbours showed that 46
they belonged to distinct species. The two isolates were readily distinguished from one 47
another and from the type strains of the other species classified in the genus Streptomyces 48
based on a combination of phenotypic and genotypic properties. Based on the genotypic and 49
phenotypic evidence, strains BNT558T and SM3501
T belong to two novel species in the genus 50
Streptomyces for which the names Streptomyces iconiensis sp. nov. (type strain BNT558T
= 51
KCTC 29198T = DSM 42109
T) and Streptomyces smyrnaeus sp. nov. (type strain SM3501
T = 52
KCTC 29214T = DSM 42105
T) are proposed, respectively. 53
Saline lakes and man-made marine salterns are interesting model systems for microbiologist 54
to work on microbial diversity and ecosystem functions in extreme environments. Recent 55
studies have shown that such environments are highly productive and have been a valuable 56
source of novel microorganisms (Anton et al., 2002; Yoon et al., 2002; Li et al., 2004; Tang 57
et al., 2010 & 2011; Wang et al., 2011; Yang et al., 2012; Tatar et al., 2013). While new 58
advances have been slow, there is great interest in the use of halophilic or halotolerant 59
microorganisms to degrade organic pollutants and to produce biopolymers, biosurfactants and 60
compatible solutes (Marhuenda-Egea & Bonete, 2002; Le Bornge et al., 2008). In this 61
context, members of the genus Streptomyces remain an unique source of natural products, 62
including clinically significant antibiotics, antimetabolities and antitumour agents (Watve et 63
al., 2001; Igarashi et al., 2005; Fiedler et al., 2005; Shiomi et al., 2005; Olano et al., 2009; 64
Kim et al., 2012a). Another unique feature of the genus is the large number of species it 65
contains; at the time of writing the genus Streptomyces contained over 600 species with 66
validly published names (Euzéby, 2012; http://www.bacterio.cict.fr/s/streptomycesa.html). 67
The present investigation was designed to establish the taxonomic positions of two novel 68
Streptomyces strains, designated BNT558T and SM3501
T, that were isolated from Tuz (Salt) 69
Lake and Camalti Saltern, in Turkey. A polyphasic taxonomic study based on a combination 70
of genotypic and phenotypic procedures showed that isolates BNT558T and SM3501
T should 71
be recognized as two novel species of the genus Streptomyces, Streptomyces iconiensis sp. 72
nov. and Streptomyces smyrnaeus sp. nov., respectively. 73
Strain BNT558T was isolated from soil samples collected from Tuz (Salt) Lake, Konya, 74
Turkey, by using Modified Bennett’s agar (Jones, 1949), supplemented with 5 % (w/v) NaCl 75
while strain SM3501T was isolated by using SM3 medium (Tan et al., 2006) supplemented 76
with filter sterilized cycloheximide (50 µg ml-1
) and 15 % (w/v) NaCl from a sediment sample 77
collected from the first pool of Camalti Saltern, Izmir, Turkey. The isolation plates were 78
incubated at 28˚C for 21 days. The strains BNT558T and SM3501
T were maintained on 79
modified Bennett’s agar slopes and on starch-mineral salt agar supplemented with 10 % (w/v) 80
NaCl (DSMZ medium 1240), respectively, at room temperature and in 20 % (v/v) glycerol 81
solution at -20 °C. 82
Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA gene and 83
purification of the PCR products were carried out following Chun & Goodfellow (1995). The 84
almost complete 16S rRNA gene sequences of strains BNT558T and SM3501
T were 85
determined using an ABI PRISM 3730 XL automatic sequencer. The resultant 16S rRNA 86
gene sequences were aligned with corresponding sequences of representative type strains of 87
the genus Streptomyces (retrieved from EzTaxon-e server; Kim et al., 2012b) by using 88
CLUSTAL W in MEGA version 5.0 (Tamura et al., 2011). The sequence similarities were 89
calculated using PHYDIT program version 3.1. Phylogenetic analysis was carried out by 90
using three tree-making algorithms: neighbour-joining (Saitou & Nei, 1987), maximum-91
likelihood (Felsenstein, 1981) and maximum parsimony (Fitch, 1971) with MEGA version 92
5.0 (Tamura et al., 2011). Evolutionary distances were calculated using model of Jukes & 93
Cantor (1969). Topologies of the resultant trees were evaluated by bootstrap analyses 94
(Felsenstein, 1985) based on 1000 resamplings. 95
Almost complete 16S rRNA gene sequences of strains SM3501T (1494 bp) and BNT558
T 96
(1473 bp), were determined. These sequences were analysed by preliminary comparison with 97
sequences in the GenBank database and the results indicated that the two isolates were most 98
closely related to members of the genus Streptomyces. It is apparent from Fig. 1 that strain 99
BNT558T and SM3501
T separated to two different subclades and shared 97.14 % 16S rRNA 100
similarity, which corresponds to 42 nt differences at 1471 locations. This relationship was 101
supported by all the tree-making algorithms used in this study (Supplementary Fig. S1 and 102
S2). The 16S rRNA gene sequence similarity between strain BNT558T and the most closely 103
related type strains Streptomyces albiaxialis NRRL B-24327T, Streptomyces daliensis YIM 104
31724T, Streptomyces sclerotialus DSM 43032
T, Streptomyces rimosus subsp. rimosus ATCC 105
10970T, Streptomyces ramulosus NRRL B-2714
T, Streptomyces olivaceiscleroticus DSM 106
40595T, and Streptomyces niger NBRC 13362
T were 98.91 % (16 nt differences at 1471), 107
98.37 % (24 nt differences at 1471), 98.15 % (27 nt differences at 1461), 98.10 % (28 nt 108
differences at 1471), 98.02 % (29 nt differences at 1466), 98.02 % (29 nt differences at 1465) 109
and 98.02 % (29 nt differences at 1464), respectively. Sequence similarities with all other type 110
strains of the genus Streptomyces were below 98.0 %. 111
Strain SM3501T
was most closely related to Streptomyces cacaoi subsp. cacaoi NBRC 112
12748T. The two strains share 98.43 % 16S rRNA sequence similarity, which corresponds to 113
23 nt differences at 1464. High sequence similarity values were also shown with the type 114
strains of Streptomyces qinglanensis 172205T (98.37 %; 24 nt differences at 1472), 115
Streptomyces flocculus NBRC 13041T (97.95 %; 30 nt differences at 1463), Streptomyces 116
rangoonensis LMG 20295T (97.82 %; 32 nt differences at 1465), Streptomyces gibsonii 117
NBRC 15415T (97.81 %; 32 nt differences at 1464), Streptomyces almquistii NRBC 13015
T 118
(97.81 %; 32 nt differences at 1459), Streptomyces albus subsp. albus NRRL B-2365T (97.76 119
%; 33 nt differences at 1472), Streptomyces hygroscopicus subsp. hygroscopicus NRRL 120
2387T (97.54 %; 36 nt differences at 1463) and Streptomyces sporocinerus NBRC 100766
T 121
(97.54 %; 36 nt differences at 1463). Sequence similarities with the type strains of all other 122
species with validly published names of the genus Streptomyces were lower. 123
124
DNA-DNA relatedness values were determined between strain BNT558T and S. albiaxialis 125
DSM 41799T, and between strain SM3501
T and the type strains of S. cacaoi subsp. cacaoi 126
KCTC 9758T, S. qinglanensis DSM 42035
T and S. flocculus DSM 40327
T. DNA was isolated 127
using a French pressure cell (Thermo Spectronic) and was purified by chromatography on 128
hydroxyapatite as described by Cashion et al. (1977). DNA-DNA hybridization was carried 129
out as described by De Ley et al. (1970) under consideration of the modifications described 130
by Huss et al. (1983) using a model Cary 100 Bio UV/VIS-spectrophotometer equipped with 131
a Peltier-thermostatted 6X6 multicell changer and a temperature controller with in situ 132
temperature probe (Varian). 133
The taxonomic integrity of the test strains were supported by DNA-DNA relatedness data. 134
Strain BNT558T showed DNA relatedness values 18.4 % ± 1.2 % to S. albiaxialis DSM 135
41799T, while strain SM3501
T showed DNA relatedness values 32.8 % ± 6.3 % to S. cacaoi 136
subsp. cacaoi KCTC 9758T, 46.2 % ± 2.7 % to S. qinglanensis DSM 42035
T and 47.7 % ± 7.4 137
% to S. flocculus DSM 40327T (based on the average of duplicate determinations in 2X SSC 138
and 10 % formamide at 70 °C), below the recommended criterion of 80 % for species 139
delineation of the genus Streptomyces (Labeda, 1992). Further DDH experiments can safely 140
be omitted, because the risk to miss a DDH value above the species discrimination threshold 141
of 80 % is neglectable (below 0.03 % for strain SM3501T and below 0.25 % for strain 142
BNT558T, even when the more strict 70 % DDH threshold proposed by Wayne et al. (1987) 143
would be considered (Meier-Kolthoff et al. 2013). 144
Biomass for chemotaxonomic studies were prepared by growing strains BNT558T and 145
SM3501T in Bennett’s broth at 160 rpm for 18 days at 28 ºC; cells were harvested by 146
centrifugation, washed in distilled water, re-centrifuged and freeze-dried. Whole cell amino 147
acids and sugars were prepared according to Lechevalier & Lechevalier (1970) and analysed 148
by thin layer chromatography (Staneck & Roberts, 1974). Polar lipids were extracted and 149
analyzed by the method of Minnikin et al. (1984) as modified by Kroppenstedt & Goodfellow 150
(2006). The isoprenoid quinones were extracted and purified using the method of Collins et 151
al. (1977) and analysed by HPLC (Kroppenstedt, 1982). For extraction and analysis of 152
cellular fatty acids, physiological age of each strain was standardized by consistently choosing 153
the last quadrant streaked on Bennett’s agar plates incubated at 28 ºC for 4 days. Analysis was 154
conducted using the Microbial Identification System (MIDI) Sherlock software version 4.5 155
(method TSBA40, TSBA6 database) as described by Sasser (1990). FAME peaks were 156
analysed using soft ware version TSBA 5.0. The DNA G+C content of strains BNT558T and 157
SM3501T were determined following the procedure developed by Gonzalez & Saiz-Jimenez 158
(2005). 159
The cell wall of strains BNT558T and SM3501
T contained LL-diaminopimelic acid. Whole-160
cell hydrolysates consisted of fucose, mannose, ribose and traces of galactose and glucose for 161
strain BNT558T, galactose, glucose, ribose and a trace of mannose for strain SM3501
T. The 162
polar lipid profile of strain BNT558T consisted of diphosphatidylglycerol, 163
phosphatidylethanolamine, phosphatidylinositol, one unidentified glycolipid and one 164
unidentified aminophospholipid. Whereas strain SM3501T contained diphosphatidylglycerol, 165
phosphatidylglycerol, phosphatidylinositol, phoshphatidylethanolamine and three unidentified 166
atypical aminolipids, one unidentified aminolipid and two unidentified glycolipids (Supp. Fig. 167
S3-4). The major menaquinones of the strain BNT558T were MK-9(H6) (54.0 %) and MK-168
9(H8) (27.0 %). Minor amounts of MK-9(H2) (2.0 %), MK-9(H4) (5.0 %), MK-10(H4) (1.0 169
%), MK-10(H6) (1.0 %), MK-10(H8) (1.0 %), and some minor unidentifed components were 170
also detected. Strain SM3501T exhibited MK-9(H8) (51.0 %) and MK-9(H6) (32.0 %) as the 171
predominant menaquinones. Minor amounts of MK-9(H4) (1.0 %), MK-10(H2) (2.0 %), MK-172
10(H4) (1.0 %), MK-10(H6) (3.0 %), MK-10(H8) (4.0 %) and in addition some minor 173
unidentified components were also detected. The major cellular fatty acids were anteiso-C15:0 174
(46.52 %), anteiso-C17:0 (18.18 %), iso-C16:0 (13.31 %) and iso-C15:0 (10.0 %) for strain 175
BNT558T, and anteiso-C15:0 (37.27 %), anteiso-C17:0 (32.28 %) and iso-C16:0 (17.31 %) for 176
strain SM3501T (Supplementary Table S1). The G+C content of the DNA was 70.2 and 69.6 177
mol % for strains BNT558T and SM3501
T, respectively. 178
Cultural characteristics were investigated on media from the International Streptomyces 179
Project (ISP) agar (Shirling & Gottlieb, 1966), modified Bennett’s agar, Czapek’s agar and 180
tryptic soy agar (TSA; Difco). The degree of growth, aerial mycelium and pigmentation were 181
recorded after 14 days of incubation at 28 °C. National Bureau of Standards (NBS) Colour 182
Name Charts (Kelly, 1964) was used for determining colour designation and names. Colony 183
morphology and micromorphological properties of strains BNT558T and SM3501
T were 184
determined by examined gold coated dehydrated specimens of 30-days cultures from 185
modified Bennett’s agar and DSMZ medium 1240, respectively, using a JEOL JSM 6060 186
scanning electron microscope. Growth at different temperatures (4, 10, 28, 37, 45, 50 and 55 187
ºC), and pH 4.0–12.0 (at intervals of 1.0 pH unit), and in the presence of NaCl (0–10, 15, 20, 188
30 %; w/v) was determined on yeast extract-malt extract agar (ISP 2) (Shirling & Gottlieb, 189
1966 ). Established methods were used to determine whether the strains degraded Tween 40 190
and 80 (Nash & Krent, 1991); the remaining degradation tests were examined using methods 191
described by Williams et al. (1983). Carbon source utilization was tested using carbon source 192
utilization (ISP 9) medium (Shirling & Gottlieb, 1966) supplemented with a final 193
concentration of 1 % of the tested carbon sources. Nitrogen source utilization was examined 194
using the basal medium recommended by Williams et al. (1983) supplemented with a final 195
concentration of 0.1 % of the tested nitrogen sources. Antimicrobial activity of strains 196
BNT558T and SM3501
T to inhibit the growth Gram (+) bacteria, such as Bacillus subtilis 197
NRRL B-209, Bacillus cereus NRRL B-3711, Bacillus licheniformis NRRL B-1001, Bacillus 198
pumilus NRRL-BD 142, Liysteria monocytogenes ATCC 19117, Micrococcus luteus NRRL 199
B-1018, Staphylococcus aureus ATCC 29213 and Staphylococcus aureus NRRL B-767, and 200
Gram (-) bacteria, such as Citrobacter freundii NRRL B-2643, Escherichia coli ATCC 25922, 201
Escherichia coli MC4100, Enterobacter aerogenes NRRL B-427 and Pseudomonas 202
aeruginosa NRRL B-2679 or fungi, such as Aspergillus niger, Aspergillus parasiticus NRRL-203
465T and Candida utilis NRRL Y-900 were observed using an agar well method described by 204
Zamanian et al., (2005). The type strains S. albiaxialis DSM 41799T, S. ferralitis DSM 205
41836T, S. cacaoi subsp. cacaoi KCTC 9758
T, S. qinglanensis DSM 42035
T, S. flocculus 206
DSM 40327T and S. rangoonensis DSM 40452
T were included for comparison in all tests. 207
208
BNT558T and SM3501
T had morphological proporties consistent with their classification in 209
the genus Streptomyces. Strain BNT558T formed a brownish gray substrate mycelium and 210
dark grayish brown aerial hyphae which differentiated into open loops of warty-surfaced 211
spore on modified Bennett’s agar while strain SM3501T formed a branched white substrate 212
mycelium and white aerial hyphae which differentiated into spiral chains of smooth-surfaced 213
spores on DSMZ medium 1240, (Fig. 2 and 3). The organism grew well on ISP 2, 3, 4, 5, 6 214
and 7, and modified Bennett’s agar. Strain BNT558T produced strong reddish-brown 215
pigments on ISP 2, grayish-brown ones on ISP 3, strong brown ones on ISP 4 and dark red 216
pigments on modified Bennett’s agar, whereas strain SM3501T produced moderate yellowish-217
brown pigments on ISP 3 medium. BNT558T and SM3501
T did not produce any melanoid 218
pigments on ISP 6 and ISP 7 medium. The physiological and biochemical properties are given 219
in Table 1 and the species description. 220
The genotypic and phenotypic data presented here show that the two isolates can be 221
distinguished from one another and from the type strains of species previously classified in 222
the genus Streptomyces. Therefore, it is concluded that strains BNT558T and SM3501
T 223
represent two novel species within the genus Streptomyces, for which the names Streptomyces 224
iconiensis sp. nov., Streptomyces smyrnaeus sp. nov. are proposed. 225
226
Description of Streptomyces iconiensis sp. nov. 227
Streptomyces iconiensis (i.co.ni.en'sis. L. masc. adj. iconiensis pertaining to Iconium, the 228
present day Konya, from where the type strain was isolated). 229
Aerobic, Gram stain-positive, non-motile, non-acid-alcohol-fast actinomycete which forms a 230
brownish gray substrate mycelium and dark grayish brown aerial hyphae which differentiated 231
into open loops of warty-surfaced spores. Growth occurs at pH 5.0-12.0, and at 28-45 ºC, but 232
not at pH 4.0 and at temperatures of 4, 10, 50 and 55 ºC. Arbutin, allantoin and urea are 233
hydrolysed. Nitrate reduction is negative. Adenine, starch, Tweens 40 and 80 are degraded but 234
casein and xanthine are not. Utilizes adonitol, D-arabinose, L-arabinose, D-cellobiose, D-235
fructose, D-galactose, D-mannitol, D-mannose, lactose, maltose, sucrose, dextrin and xylose 236
as sole carbon sources, but not D-sorbitol, dextran, inulin, L-sorbose, L-glutamic acid, xylitol 237
and succinic acid. Utilizes glycine, L-alanine, L-arginine, L-methionine, L-serine as sole 238
nitrogen sources, but not alpha-iso-leucine, L-phenylalanin, L-histidine, L-threonine, L-proline, 239
L-hydroxyproline, L-cysteine, L-valine. Antimicrobial activity is shown against Aspergillus 240
parasiticus NRRL-465, Candida utilis NRRL Y-900, Escherichia coli MC4100, Bacillus 241
subtilis NRRL B-209, Bacillus pumilus NRRL-BD 142, Staphylococcus aureus ATCC 29213, 242
Staphylococcus aureus NRRL B-767 The predominant menaquinones were MK-9(H6) and 243
MK-9(H8). Major fatty acids were anteiso-C15:0, anteiso-C17:0 and iso-C16:0. The polar lipid 244
profile contains diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, one 245
unidentified glycolipid and one unidentified aminophospholipid. The G+C content of the 246
genomic DNA was 70.2 mol %. The type strain, BNT558T (= KCTC 29198
T = DSM 42109
T) 247
was isolated from a soil sample collected from Tuz (Salt) Lake, Konya, Turkey. 248
249
250
251
Description of Streptomyces smyrnaeus sp. nov. 252
Streptomyces smyrnaeus (smyr.nae’us. L. masc. adj. smyrnaeus pertaining to Smyrna, the 253
present-day Izmir, from where the type strain was isolated) 254
Aerobic, Gram stain-positive, non-motile, non-acid-alcohol-fast actinomycete which forms a 255
branched white substrate mycelium and white aerial hyphae which differentiated into spiral 256
chains of smooth-surfaced spores. Growth occurs at pH 4.0-12.0, and at 28-45 ºC, but not at 257
temperatures of 4, 10, 50 and 55 ºC. Arbutin, allantoin are hydrolysed, but not urea. Nitrate 258
reduction is negative. Adenine, starch, Tweens 40 and 80 are degraded but casein and 259
xanthine are not. Utilizes adonitol, L-arabinose, D-cellobiose, D-fructose, D-galactose, D-260
sorbitol, D-mannitol, D-mannose, lactose, maltose, sucrose, dextrin, inulin, xylitol, xylose and 261
succinic acid as sole carbon sources, but not D-arabinose, dextran, L-sorbose and L-glutamic 262
acid. Utilizes alpha-iso-leucine, glycine, L-alanine, L-arginine, L-hydroxyproline, L-threonine, 263
L-proline, L-serine, L-valine as sole nitrogen sources, but not L-phenylalanin, L-methionine, 264
L-histidine, L-cysteine. Antimicrobial activity is shown against Aspergillus parasiticus 265
NRRL-465, Candida utilis NRRL Y-900, Bacillus subtilis NRRL B-209, Bacillus cereus 266
NRRL B-3711, Bacillus pumilus NRRL-BD 142. The predominant menaquinones were MK-267
9(H8) and MK-9(H6). Major fatty acids of the strains were anteiso-C15:0, anteiso-C17:0 and iso-268
C16:0. The polar lipid profile contains diphosphatidylglycerol, phosphatidylglycerol, 269
phosphatidylinositol, phoshphatidylethanolamine and three unidentified atypical aminolipids, 270
one unidentified aminolipid and two unidentified glycolipids, The G+C content of the 271
genomic DNA was 69.6 %. The type strain, SM3501T(= KCTC 29214
T = DSM 42105
T) was 272
isolated from a sediment sample collected from first pool of Camalti Saltern, Izmir, Turkey. 273
274
Acknowledgements 275
This research was supported by Anadolu University (AU), project no. 1201F012. 276
References 277
Antón, J., Oren, A., Benlloch, S., Rodríguez-Valera, F., Amann, R. & Rosselló-Mora, R. 278
(2002). Salinibacter ruber gen. nov., sp. nov., a novel, extremely halophilic member of the 279
Bacteria from saltern crystallizer ponds. Int J Syst Evol Microbiol 52, 485–491. 280
Cashion, P., Holder-Franklin, M. A., McCully, J. & Franklin, M. (1977). A rapid method 281
for the base ratio determination of bacterial DNA. Anal Biochem 81, 461–466. 282
Chun, J. & Goodfellow, M. (1995). A phylogenetic analysis of the genus Nocardia with 16S 283
rRNA gene sequences. Int J Syst Bacteriol 45, 240–245. 284
Collins, M.D., Pirouz, T., Goodfellow, M. & Minnikin, D.E. (1977) Distribution of 285
menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100, 221–230. 286
De, Ley. J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA 287
hybridization from renaturation rates. Eur J Biochem 12, 133–142. 288
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood 289
approach. J Mol Evol 17, 368–376. 290
Felsenstein, J. (1985). Confidence limits on phylogeny: an approach using the bootstrap. 291
Evolution 39 783–791. 292
Fiedler, H.-P., Bruntner, C., Bull, A. T., Ward, A. C., Goodfellow, M., Potterat, O., 293
Puder, C. & Mihm, G. (2005). Marine actinomycetes as a source of novel secondary 294
metabolites. Antonie van Leeuwenhoek 87, 37–42. 295
Fitch, W. M. (1971). Toward defining the course of evolution: minimum change for a 296
specific tree topology. Syst Zool 20, 406–416. 297
298
Gonzalez, J. M. & Saiz-Jimenez, C. (2005). A simple fluorimetric method for the estimation 299
of DNA-DNA relatedness between closely related microorganisms by thermal denaturation 300
temperatures. Extremophiles 9, 75–79. 301
Huss, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the spectrophotometric 302
determination of DNA hybridization from renaturation rates. Syst Appl Microbiol 4, 184–192. 303
Igarashi,M., Takahashi, Y., Shitara, T., Nakamura, H., Naganawa, H., Miyake, T., et al. 304
(2005). Caprazamycins, novel lipo-nucleoside antibiotics, from Streptomyces sp. II. Structure 305
elucidation of caprazamycins. J Antibiot 58 327–337. 306
Jones, K. L. (1949). Fresh isolates of actinomycetes in which the presence of sporogenous 307
aerial mycelia is a fluctuating characteristic. J Bacteriol 57 141–145. 308
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein 309
Metabolism vol. 3, pp. 21–132. Edited by H. N. Munro. New York: Academic Press 310
Kelly, K. L. (1964). Inter-Society Color Council-National Bureau of Standards color-name 311
charts illustrated with centroid colors published in US. 312
Kim, B.Y., Zucchi, T.D., Fiedler, H.P. & Goodfellow, M. (2012a). Streptomyces cocklensis 313
sp nov., a dioxamycin-producing actinomycete. Int J Syst Evol Microbiol, 62, 279–283. 314
Kim, O.-S., Cho, Y.-J., Lee, K., Yoon, S.-H., Kim, M., Na, H., Park, S.-C., Jeon, Y. S., 315
Lee, J. H., Yi, H., Won, S. & Chun, J. (2012b). Introducing EzTaxon-e: a prokaryotic 16S 316
rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst 317
Evol Microbiol 62, 716–721. 318
Kroppenstedt, R. M. (1982). Separation of bacterial menaquinones by HPLC using reverse 319
phase (RP-18) and a silver-loaded ion exchanger. J Liq Chromatogr 5, 2359–2367. 320
Kroppenstedt, R.M. & Goodfellow, M. (2006). The family Thermomonosporaceae: 321
Actinocorallia, Actinomadura, Spirillispora and Thermomonospora. In: Dworkin M, Falkow 322
S,Schleifer KH, Stackebrandt E (eds) The prokaryotes, archaea and bacteria: firmicutes, 323
actinomycetes, 3rd edn, vol 3. Springer, New York, pp 682–724 324
Labeda, D. P. (1992). DNA–DNA hybridization in the systematics of Streptomyces. Gene 325
115, 249–253. 326
Le Borgne S, Paniagua D. & Vazquez-Duhalt R. (2008) Biodegradation of organic 327
pollutants by halophilic bacteria and archaea. J Mol Microbiol Biotechnol 15, 74–92. 328
Lechevalier, M. P. & Lechevalier, H. (1970). Chemical composition as a criterion in th e 329
classification of aerobic actinomycetes. Int J Syst Bacteriol 20, 435–443. 330
Li, W.-J., Park, D.-J., Tang, S.-K., Wang, D., Lee, J.-C., Xu, L.-H., Kim, C.-J. & Jiang, 331
C.-L. (2004). Nocardiopsis salina sp. nov., a novel halophilic actinomycete isolated from 332
saline soil in China. Int J Syst Evol Microbiol 54, 1805–1809. 333
Marhuenda-Egea, F.C. & M.J. Bonete, (2002). Extreme halophilic enzymes in organic 334
solvents. Curr Opin Biotechnol 13, 385–389. 335
Meier-Kolthoff, J. P., Göker, M., Spröer, C., Klenk, H.-P. (2013). When should a DDH 336
experiment be mandatory in microbial taxonomy? Arch Microbiol 195, 413-418. 337
Minnikin, D. E., O'Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, K. 338
& Parlett, J. H. (1984). An integrated procedure for the extraction of bacterial isoprenoid 339
quinones and polar lipids. J Microbiol Methods 2, 233–241. 340
Nash, P. & Krent, M. M. (1991). Culture media. In Manual Of Clinical Microbiology, 5th 341
Edition, pp: 1268–1270, Edited by A. Ballows, W.J. Hauser, K.L. Herrmann, H.D. Isenberg & 342
H.J. Shadomy. American Society for Microbiology, Washington DC. 343
Olano, C., Gómez, C., Pérez, M., Palomino, M., Pineda-Lucena, A., Carbajo, R. J., 344
Braňa, A. F., Méndez, C. & Salas, J. A. (2009). Deciphering biosynthesis of the RNA 345
polymerase inhibitor streptolydigin and generation of glycosylated derivatives. Chem Biol 16, 346
1031–1044. 347
Saitou, N. & Nei, M. (1987). The neighbor-joining method. A new method for reconstructing 348
phylogenetic trees. Mol Biol Evol 4, 406–425. 349
Sasser, M. (1990). Identification of Bacteria By Gas Chromatography of Cellular Fatty 350
Acids. Technical Note 101. Newark, DE: MIDI Inc. 351
Shiomi, K., K. Hatae, H. Hatano, A. Matsumoto, Y. Takahashi, C.L. Jiang, H. Tomoda, 352
S. Kobayashi, H. Tanaka, and S. Omura. (2005). A new antibiotic, antimycin Ag, produced 353
by Streptomyces sp. K01-0031. J. Antibiot, 58, 74–78. 354
Shirling, E. B. & Gottlieb, D. (1966). Methods for characterization of Streptomyces species, 355
Int J Syst Bacteriol 16, 313–340. 356
Staneck, J. L. & Roberts, G. D. (1974). Simplified approach to identification of aerobi c 357
actinomycetes by thin-layer chromatography. Appl Microbiol 28, 226–231. 358
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). 359
MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, 360
Evolutionary Distance, and Maximum Parsinomy Methods. Mol Bio Evol 28, 2731–2739. 361
Tan, G. Y. A., Ward, A. C. & Goodfellow, M. (2006). Exploration of Amycolatopsis 362
diversity in soil using genus-specific primers and novel selective media. Syst Appl Microbiol 363
29, 557–569. 364
Tang, S.K., Wang, Y., Guan, T,W., Lee, J.C., Kim, C.J. & Li, W.J. (2010). Amycolatopsis 365
halophila sp. nov., a halophilic actinomycete isolated from a salt lake. Int J Syst Evol 366
Microbiol 60,1073–1078. 367
Tang, S.K., Zhi, X.Y., Wang, Y., Shi, R., Lou, K., Xu, L.H., Li, W.J. (2011). 368
Haloactinopolyspora alba gen. nov., sp. nov., a halophilic filamentous actinomycete isolated 369
from a salt lake, with proposal of Jiangellaceae fam. nov. and Jiangellineae subord. nov. Int J 370
Syst Evol Microbiol 61, 194–200. 371
Tatar, D., Sazak, A., Guven, K., Sahin, N. (2013). Amycolatopsis cihanbeyliensis sp. nov., a 372
halotolerant actinomycete isolated from Lake Salt in Turkey. Int J Syst Evol Microbiol. 63, 373
3739–3743 374
Wang, Y., Tang, S.K., Li, Z., Lou, K., Mao, P.H., Jin, X., Klenk, H.P., Zhang, L.X. & Li, 375
W.J. (2011). Myceligenerans halotolerans sp. nov., an actinomycete isolated from a salt lake, 376
and emended description of the genus Myceligenerans. Int J Syst Evol Microbiol 61, 974–978. 377
Watve, M.G., Tickoo, R., Jog, M.M. & Bhole, B.D. (2001). How many antibiotics are 378
produced by the genus Streptomyces? Arch Microbiol 176, 386–390. 379
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., 380
Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E., Stackebrandt, E., 381
Starr, M. P. & Trueper, H. G. (1987). Report of the Ad Hoc committee of reconciliation of 382
approaches to bacterial systematics. Int J Syst Bacteriol 37, 463-464. 383
Williams, S. T., Goodfellow, M., Alderson, G., Wellington, E. M. H., Sneath, P. H. A. & 384
Sackin, M. J. (1983). Numerical classification of Streptomyces and related genera. J Gen 385
Microbiol 129, 1743–1813. 386
Yang, C.X., Liu, Y.P., Bao, Q.H., Feng, F.Y., Liu, H.R., Zhang, X.J. & Zhao, Y.L. 387
(2012). Mongoliitalea lutea gen. nov., sp. nov., an alkaliphilic, halotolerant bacterium isolated 388
from a haloalkaline lake. Int J Syst Evol Microbiol 62, 647–653. 389
Yoon, J.-H., Lee, K.-C., Kho, Y. H., Kang, K. H., Kim, C.-J. & Park, Y.-H. (2002). 390
Halomonas alimentaria sp. nov., isolated from jeotgal, a traditional Korean fermented 391
seafood. Int J Syst Evol Microbiol 52, 123–130. 392
Zamanian, S., G.H. Shahidi Bonjar and I. Saadoun, (2005). First report of antibacterial 393
properties of a new strain of Streptomyces plicatus (strain 101) against Erwinia carotovora 394
from Iran. Biotechnology 4, 114–120. 395
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Table 1. Phenotypic properties of strains BNT558T, SM3501T and the most related type species. Strains: 1, BNT558; 2, 415
Streptomyces albiaxialis DSM 41799T; 3, Streptomyces ferralitis DSM 41836T; 4, SM3501; 5, Streptomyces cacaoi subsp. 416
cacaoi KCTC 9758T; 6, Streptomyces qinglanensis DSM 42035T; 7, Streptomyces flocculus DSM 40327T; 8, Streptomyces 417
rangoonensis DSM 40452T.Strains were positive for arbutin hydrolysis, ability of growth at D-fructose, D-galactose, D-418
mannose, D-mannitol, dextrin, lactose, maltose, xylose as sole carbon sources 1.0 % (w/v), L-alanine and L-arginine as sole 419
nitrogen sources 0.1 % (w/v), growth at pH:6.0, pH:8.0, pH:10, temperatures 28 °C, 37°C, 45°C and 0-5 % NaCl 420
concentrations. But negative for ability of growth at dextran, L-sorbose, L-glutamic acid, growth at 4°C, 10°C, 55°C and 20 421
%, 30 % NaCl concentrations . All data were obtain in this study. 422
423
1 2 3 4 5 6 7 8
Biochemical Tests
Allantoin Hydrolysis + + + + + - + +
Nitrate Reduction - - - - - - - +
Urea Hydrolysis + - - - - + + -
pH tolerence
4.0 - + - + + - + +
5.0 + + + + + - + +
11.0 + + - + + + + + 12.0 + + - + + + + +
Temperature
50°C - - - - - - + +
NaCl (%)
8.0 + + - + + + + +
9.0 + + - + + + + +
10.0 + + - + + + + + 15.0 - - - + + + - -
Degradation
Adenine (0.5%) + + - + + + - - Casein (1.0 %) - - + - - - - -
Starch (1.0 %) + + + + + + - - Tween 40 (1.0 %) + + - + + + + + Tween 80 (1.0 %) + + - + + + + +
Use of sole C sources 1.0 % (w/v)
Adonitol + + - + + + + + D-arabinose + + - - + + - -
L-arabinose + - - + + + - -
D-cellobiose + + - + + + + + D-sorbitol - - - + - - - -
Inulin - + - + + + - +
Succinic acid (0.1 %) - + - + - + + + Sucrose (Saccharose) + + - + + + + +
Xylitol - - - + - - - -
Use of sole N sources 0.1 % (w/v)
Alpha-isoleucine - + + + + + + +
Glycine + + + + + - + + L-cysteine - + + - + - + +
L-histidine - + + - + - + +
L-hydroxyproline - + + + - - - + L-methionine + + + - - - - -
L-phenylalanine - + + - - - - +
L-proline - + + + + - + + L-serine + + + + + - + +
L-threonine - + + + + + + +
L-valine - + + + + - + +
424
425
426
427
428
429
Legends for Figures 430
Fig. 1. Neighbour-joining tree (Saitou & Nei, 1987) based on almost complete 16 rRNA gene 431
sequences showing the position of strains BNT558T and SM3501
T amongst their phylogenetic 432
neighbours. Actinomadura glomerata IMSNU 22179T (AJ293704) was used as an out group. 433
Numbers at the nodes indicate the levels of bootstrap support (%); only values ≥ 50% are 434
shown. GenBank accession numbers are given in parentheses. Bar, 0.01 substitutions per site. 435
436
Fig. 2. Scanning electron micrograph of strain BNT558T grow on modified Bennett’s agar at 437
28°C for 21 days. 438
439
Fig. 3. Scanning electron micrograph of strain SM3501T grow on starch-mineral salt agar 440
supplemented with 10 % (w/v) NaCl (DSMZ medium 1240) at 28°C for 21 days. 441
442
Supplementary Fig. S1. Maximum parsimony tree based on almost complete 16 rRNA gene 443
sequences showing the position of strains BNT558T and SM3501
T amongst their phylogenetic 444
neighbours. 445
Supplementary Fig. S2. Maximum likelihood tree based on almost complete 16 rRNA gene 446
sequences showing the position of strains BNT558T and SM3501
T amongst their phylogenetic 447
neighbours. 448
449
Supplementary Fig. S3. Molybdophosporic acid stained two-dimensional TLC of polar 450
lipids from strain BNT558T. 451
452
DPG: Diphosphatidylglycerol, PE: Phosphatidylethanolamine, PI: Phosphatidylinositol, GL: 453
Glycolipid and PN: Aminophospholipid 454
Supplementary Fig. S4. Molybdophosporic acid stained two-dimensional TLC of polar 455
lipids from strain SM3501T. 456
457
DPG: Diphosphatidylglycerol, PE: Phosphatidylethanolamine, PG: Phosphatidylglycerol and 458
AL, atyp: Aminolipid, AL: Aminolipid and GL: Glycolipid. 459
460
Supplementary Table S1. Fatty acids profiles of strains BNT558T and SM3501
T and closely 461
related type species. 462
463
464
465
466
467
468
469